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Background and Objective: Hemophilia A is the most common type of this disease. For the treatment of hemophiliacs, the injectable concentrate of human factor VIII, derived from plasma and recombinant, is available in the market. Due to the risk of contracting infectious diseases due to blood transfusions or donated blood products, including plasma-driven factor VIII, recombinant factor VIII can be used, which is safe and has a higher purity than the blood-derived sample, while showing a longer half-life in the blood of consuming patients. The aim of this study is to evaluate the expression and synthesis of recombinant human factor VIII using a cell-free eukaryotic expression system as a new strategy for the synthesis of recombinant drugs. Methods: In this experimental study, pcDNA3 vector containing the gene fragment without B-domain of blood coagulation factor VIII was transformed into susceptible DH5a bacteria. After the multiplication of the bacteria and the increase of the target vector, the amplified vector was extracted at this stage and its quality and concentration were determined. Then CHO-K1 eukaryotic cells were cultured and cell extract was prepared from the density of more than ten million of their cells. The extracted vector was combined with cell extract and expression solution, and the expression process was carried out at 30°C and 37°C. The level of expression was evaluated by ELISA and dot blot techniques. Findings: Based on the ELISA technique adopted in this study, the expression level of recombinant human blood coagulation factor VIII in the base sample at a temperature of 30°C, sample 2 at a temperature of 30°C, base sample at a temperature of 37°C, and sample 4 of negative control at a temperature of 30°C were respectively reported as 275, 276, 273 and 0 ng/ml in a period of 20 hours. In the dot blot technique, the mentioned quantitative results were qualitatively confirmed by the color intensity of the spots. Conclusion: The results showed that the cell-free expression method can be used for the synthesis of recombinant factor VIII. Under the conditions used in this research for cell-free expression of recombinant factor VIII, volume ratio of 50/50 of CHO-K1 cell extract was suitable, and the basic expression solution at 30°C showed the highest concentration of the product. Due to the use of CHOK1 eukaryotic cell extract, the synthesized recombinant human factor VIII has post-translational modifications, which are very important for the functionality of this protein in the human body. [ABSTRACT FROM AUTHOR]